title: Pseudomonas putida AlkA and AlkB proteins comprise different defense systems for the repair of alkylation damage to DNA - in vivo, in vitro, and in silico studies.
abstract: Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against alkylating agents of exo- and endogenous origin.
authors.affiliation: Department of Molecular Biology, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Damian
authors.initials: D
authors.lastname: Mielecki
authors.forename: Signe
authors.initials: S
authors.lastname: Saumaa
authors.forename: Michał
authors.initials: M
authors.lastname: Wrzesiński
authors.forename: Agnieszka M
authors.initials: AM
authors.lastname: Maciejewska
authors.forename: Karolina
authors.initials: K
authors.lastname: Żuchniewicz
authors.forename: Anna
authors.initials: A
authors.lastname: Sikora
authors.forename: Jan
authors.initials: J
authors.lastname: Piwowarski
authors.forename: Jadwiga
authors.initials: J
authors.lastname: Nieminuszczy
authors.forename: Maia
authors.initials: M
authors.lastname: Kivisaar
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Grzesiuk
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000478:Alkylation; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016000:Cluster Analysis; D016384:Consensus Sequence; D004249:DNA Damage / Q000235:genetics*; D045647:DNA Glycosylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics; D059372:Nucleotide Motifs; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D000477:Alkylating Agents; D001426:Bacterial Proteins; D045647:DNA Glycosylases; C076131:DNA-3-methyladenine glycosidase II